Synapse formation and subsequent maturation involves complex interactions between the presynaptic cell and its postsynaptic target. As a model synapse, the neuromuscular junction (NMJ) provides a relatively simple system to study molecular mechanisms mediating some of these interactions. At this synapse, synaptic transmission between the motor neuron and its target muscle fiber is mediated by the muscle nicotinic acetylcholine receptor (nAChR), a multisubunit ligand-gated ion channel (Hall, Z. W., and Sanes, J. R. (1993) Cell 10 (Suppl.) 99-121).
During the development of the neuromuscular synapse, nerve-evoked muscle activity suppresses expression of embryonic-type nAChR (.alpha..sub.2 .beta..gamma..delta.) throughout the muscle fiber (Hall, Z. W., and Sanes, J. R. (1993) Cell 10 (Suppl.) 99-121; Goldman, D., et al. (1988) Neuron 1, 329-333) while muscle innervation induces expression of adult-type nAChRs (.alpha..sub.2 .beta..epsilon..delta.) at the endplate. This spatially restricted expression of adult-type nAChRs is largely a result of selective induction of the genes encoding these subunits in endplate-associated myonuclei (Hall, Z. W., et al. (1993) Cell 10 (Suppl.) 99-121). The transcriptional mechanisms by which the motor neuron regulates gene expression in these subsynaptic nuclei are not well understood.
Protein-tyrosine phosphatase (PTPase) activity selectively suppresses muscle adult-type nAChR genes. Changes in protein tyrosine phosphorylation have been proposed to contribute to synapse-specific gene expression (Sapru, M. K., et al. (1994) J. Biol. Chem. 269, 27811-27814). Neuregulins are motor neuron-derived factors that function as ligands for EGF receptor-related (erbB) tyrosine kinases and stimulate adult-type nAChR synthesis (Bargmann, C. I., et al. (1986) Nature 319, 226-230; Falls, D. L., et al. (1993) Cell 72, 801-815; Martinou, J. C., et al. (1991) Proc. Natl. Acad. Sci. USA 88, 7669-7673; Marchionni, M. A., et al. (1993) Nature 362, 312-317; Altiok, N., et al. (1995) EMBO J. 14, 4258-4266; Jo, S. A., et al. (1995) Nature 373, 158-161; Zhu, X. J., et al. (1995) EMBO J. 14, 5842-5848; Chu, G. C., et al. (1995) Neuron 14, 329-339). Neuregulins include neu differentiation factor, heregulin, glial growth factor, and acetylcholine receptor inducing activity (ARIA), which are alternatively spliced products of a single gene (Marchionni, M. A., et al. (1993) Nature 362, 312-317; U.S. Pat. No. 5,237,056; WO 94/08007). Whether the neuregulin signaling pathway differs from that perturbed by PTPase overexpression is not known.
Since the .epsilon.-subunit gene is unique to adult-type nAChRs and is locally expressed at the endplate, it serves as a marker for regulatory mechanisms involved in synapse-specific expression. In addition, this gene is most sensitive to neuregulin induction (Martinou, J. C., et al. (1991) Proc. Natl. Acad. Sci. USA 88, 7669-7673). Si et al. (Si., J. et al. (1997) J. Biol. Chem. 272:10, 367-10, 371) have recently identified a 10 nucleotide element in the mouse .epsilon.-subunit promoter (-55 to -46 upstream of the start site of transcription in the mouse) that is responsive to ARIA (ARE). It shares no significant identity with the NRE of the present invention, and is approximately 50 nucleotides from the 5' end of a mouse sequence that is identical to rat NRE in 13 of 15 nucleotides. However, a regulatory element in the .epsilon.-promoter conferring responsiveness to neuregulin, responsiveness to activated Ras, and responsiveness to PTPase has not been identified.